The present invention relates to the detoxification of bacterial endotoxin (lipopolysaccharide) by enzymatic modification of its lipid A moiety. The prophylactic and therapeutic use of such detoxified endotoxins is also a subject of the present invention.
Animals mount a complex array of inflammatory responses to tissue invasion by gram-negative bacteria. Many of these responses appear to be provoked by the lipopolysaccharides (LPS) that are present in the bacterial outer membrane. Much evidence suggests that the lipid A region of LPS directly stimulates host cells such as macrophages, neutrophils, and endothelial cells, which then mediate the inflammatory changes. Responses to LPS may be toxic (hypotension, coagulation disturbances, death) or beneficial to the infected host (enhancement of antibody formation, mobilization of phagocytes, acute phase protein synthesis, others).
Typical gram-negative bacterial lipopolysaccharides (LPS) have 3 major structural regions: the O polysaccharide, the R-core oligosaccharide, and lipid A (FIG. 1 and FIG. 2). The structure of the O polysaccharide is highly variable between organisms, even in the same species, and its antigenicity serves as a basis for serotyping the bacteria. The R region is a bridge between the O-antigen and lipid A; its structure is similar in most gram-negative bacteria. Antibodies to LPS (typically directed to O- or R-core antigenic sites) may promote phagocytosis or killing of the bacteria, or they may enhance removal of LPS from the bloodstream into sites (liver, spleen) where the LPS are degraded. The O-antigen is the most antigenic component of the LPS, yet it has little known toxicity. Lipid A, in contrast, is poorly antigenic but contains the toxic center of the molecule. The lipid A moiety is remarkably similar in structure across a wide range of bacterial genera.
The lipid A of enteric bacteria (e.g. Salmonella, E. coli) is a glucosamine disaccharide that is phosphorylated at positions 1 and 4' and has 6 or 7 covalently-linked fatty acids (FIG. 1). Four molecules of 3-hydroxytetradecanoate (3-OH-14:0) are attached to the glucosamine disaccharide at positions 2, 3, 2', and 3'; the hydroxyl groups of the 3-OH-14:0 residues at positions 2' and 3' (and sometimes 2) are substituted with normal fatty acids (dodecanoate, tetradecanoate, hexadecanoate) to form acyloxyacyl groups. In 1983 the discovery of a novel enzymatic activity was reported. This enzyme was an acyloxyacyl hydrolase, found in the granule fraction of human peripheral blood neutrophils, that selectively removed the nonhydroxylated acyl chains from Salmonella typhimurium LPS (Hall and Munford (1983) Proc. Nat. Acad. Sci. V 80, pp 6671-6675). It was known that Dictyostelium discoideum (slime mold), which utilizes Gram-negative bacteria as a major foodstuff, contains enzymes that remove nonhydroxylated and hydroxylated acyl chains from LPS (Rosner et al. (1979) J. Biol. Chem. V 254, pp 5926-5933). The experiments of Gimber and Rafter (Arch. Biochem. Biophys, (1969), V 135, pp 14-20) had also suggested that deacylation of LPS is carried out by intact neutrophils.
Recent studies of the biological activities of chemically synthesized lipid A analogs and biosynthetic precursors of lipid A ((Galanos et al. (1984) Eur J. Biochem. V 140, p 221; Takada et al. (1985) Infect. & Immun. V 48, p 219; Kotani et al. (1985) Infect. I Immun. V 49, p 225; and Homma et al. (1985) J. Biochem. V 98, p 395)) have provided valuable information about structure-activity relationships. Lipid A analogs that lack nonhydroxylated acyl chains are not reactive in the dermal Shwartzman test and have reduced pyrogenicity, yet they are nearly equipotent with complete lipid A in various assays of immune stimulation (such as B-cell mitogenicity, adjuvanticity, and stimulation of macrophages to release PGE.sub.2). The effects of acyloxyacyl hydrolase on the biological activities of LPS are in general agreement with these earlier findings: acyloxyacyl hydrolysis detoxifies LPS without destroying the immunostimulatory activity.